Ultrasound-mediated high-speed biological reaction and tissue processing

ABSTRACT

Methods of fixing and processing tissue and samples on a membrane by using ultrasound radiation as a part of the method are presented. Ultrasound of a frequency in the range of 0.1-50 MHz is used and the sample or tissue receives 0.1-200 W/cm 2  of ultrasound intensity. The use of ultrasound allows much shorter times in the methods. Also presented are apparati comprising transducers of one or of multiple heads for producing the ultrasound radiation and further comprising a central processing unit and optionally comprising one or more sensors. Sensors can include those to measure and monitor ultrasound and temperature. This monitoring system allows one to achieve accurate and optimum tissue fixation and processing without overfixation and tissue damage. The system also allows the performance of antigen-antibody reactions or nucleic acid hybridizations to be completed in a very short time while being highly specific and with a very low or no background.

BACKGROUND OF THE INVENTION

The process of fixation forms the foundation for the preparation oftissue sections. It prevents or arrests autolysis and putrefaction,coagulates and stabilizes soluble and structural proteins, fortifies thetissues against the deleterious effects of subsequent processing andfacilitates staining. Current methods of fixation rely on chemicalagents, the most widely used being formaldehyde. Although autolysis isknown to be retarded by cold and almost inhibited by heating to 60° C.(Drury and Wallington, 1980), heat as a form of tissue fixation has notbeen exploited in the diagnostic laboratory.

The use of routinely fixed, paraffin-embedded tissue sections forimmunohistochemistry staining permits localization of a wide variety ofantigens while retaining excellent morphologic detail. However, mostchemical fixatives produce denaturation or masking of many antigens anddegradation of RNA and DNA. In fact for some antigens, treatment offixed tissue sections with proteases is required for their demonstration(Brandzaeg, 1982; Taylor, 1986). Furthermore, the introduction ofantigen retrieval by heating tissue sections in a microwave oven (Shi etal., 1991) or pressure cooker (Norton et al., 1994; Miller et al., 1995)before immunostaining has been a major breakthrough in improving aresult of no or weak immunoreactivity, particularly in suboptimallyprepared tissue. However, while the optimal time for fixation varieswith the chemical agent employed, this generally takes hours,approximately a day, to accomplish.

Fixative type and fixation time are known to influence 1) thepreservation of tissue morphology (Baker, 1959), 2) the preservation ofprotein antigens for IHC (Williams et al., 1997), and 3) thepreservation of nucleic acids for ISH (Weiss and Chen, 1991; Nuovo andRichart, 1989) and PCR (Ben-ezra et al., 1991). It is fortunate thatformalin was found to be the best fixative for meeting these threecriteria, as this is the fixative most commonly used in routine tissuefixation (Weiss and Chen, 1991; Williams et al., 1997; Nuovo andRichart, 1989).

Increasing the speed and reducing the time of fixation have beeninvestigated using treatments of cold, heat, vacuum, microwave,ultrasound, and microwave combined with ultrasound. Tissues have beenmicrowave irradiated for less than 10 seconds in the presence ofchemical cross-linking agents (final solution temperature of 45-70° C.)(Login and Dvorak, 1985; Login et al., 1987). These MW fixation methodsused heat to Pasteurize the tissue rather than to fix the tissue. The 10seconds was not enough time to allow even penetration and completereaction with the tissues. Also, 70° C. is not hot enough to inhibit allenzymes such as RNases (Sambrook et al., 1989). Furthermore, Azumi andBattifora reported that the improvement seen in antigen preservation inMW fixed tissues was not due to the microwave irradiation per se butrather to the graded alcohol dehydration steps in the tissue processor(Azumi et al. 1990). The exact amount of MW energy received by tissuewas very difficult to control (Login and Dvorak, 1985; Azumi et al.,1990). Therapeutic ultrasound (800-880 kHz frequency and 1.4-2 W/cm²intensity) did not significantly improve the quality and time offixation (Drakhli, 1967; Botsman and Bobrova, 1968; Obertyshev, 1987;Rozenberg, 1991) even when combined with MW energy (Shmurun, 1992).Cleaning ultrasound (destructive low frequency 40 kHz) was also usedwith MW. Disruptions, fissures and cracks of tissues treated for only 3seconds with ultrasound irradiation (X 45 cycles) were observed when thespecimens were examined by light microscopy (Yasuda et al., 1992). Ourfindings are the same as those reports that low frequency ultrasoundexposure can lead to destruction of cell and tissue structure. Thisindicates that the safety range of low frequency ultrasound isrelatively narrow.

In the past decade, molecular pathology has been rapidly developed byusing new techniques such as immunohistochemistry (IHC), in situhybridization (ISH), fluorescent in situ hybridization (FISH),polymerase chain reaction (PCR), reverse transcription (RT)-PCR, and insitu-PCR. The most advanced techniques such as laser capturemicrodissection (LCM) (Emmert-Buck et al., 1996; Bonner et al., 1997;Fend et al., 1999a; Fend et al., 1999b), cDNA (Schena et al., 1995;DeRisi et al., 1996) and tissue microarrays (Kononen et al. (1998) havebeen developed for research and diagnosis of molecular pathology. Manygenes and signaling pathways controlling cell proliferation, death anddifferentiation, as well as genomic integrity, have been measured bythese techniques in a single experiment, revealing many new, potentiallyimportant cancer genes. However, the tissue blocks or sections used foranalysis of molecular information of LCM and tissue microarraytechniques have not fitted well with the classic method of tissuefixation—formalin fixed paraffin embedded (FFPE) tissue, which hasprovided the best morphology for pathologists throughout this century(Fend et al., 1999; Goldsworthy et al., 1999).

FFPE tissues have been extensively studied during the last two decadesfor molecular biology and molecular pathology. There have been manybreakthroughs in these areas such as success in isolating the 1918“Spanish” influenza virus RNA from an 80 year old FFPE tissue block(Taubenferger et al., 1997). However, there are many drawbacks in usingFFPE tissue for molecular pathology, such as inconsistency in fixationcondition, antigen masking, and RNA/DNA degradation. Even using theadvanced microwave-antigen retrieval method (Shi et al., 1991), severalCD markers have not worked with FFPE tissues, and the average length ofRT-PCR products from FFPE tissues is 200 bp (Fend et al., 1999a;Ben-ezra et al., 1991; Foss et al., 1994; Krafft et al., 1997). Allthese drawbacks limit the use of LCM and tissue microarray techniqueswith FFPE tissues (Fend et al., 1999a; Goldsworthy et al., 1999).

Six to eighteen hours are required for routine fixation of surgicaltissue specimens. Eight to fourteen hours are required for tissueprocessing. Additional times are required for embedding, sectioning,staining, and coverslipping of the specimen. A method whichsimultaneously permits rapid tissue fixation and processing, excellentmorphologic detail, antigen preservation, and less RNA/DNA degradationwould, therefore, be highly desirable in this molecular pathology era.

For the past three decades, microwave (MW) energy has sometimes beenused for rapid tissue fixation (Mayers, 1970; Bernard, 1974; Login,1978) and tissue processing (Boon et al., 1986) for light and electronmicroscopy. In the late 60's to early 90's, several Russian groupsdescribed a method in which therapeutic ultrasound (US) energy was usedfor tissue fixation and processing for light microscopy (Drakhli, 1967;Botsman and Bobrova, 1968; Obertyshev, 1987; Rozenberg, 1991) and forelectron microscopy (Polonyi et al., 1984; Robb et al., 1991). MW energycombined with US energy was used in conjunction with chemicalcross-linking agents to fix and process tissues for light (Shmurun,1992) and for electron microscopy (Yasuda et al., 1992) at the sametime. However, these technologies have not been successfully adopted inclinical diagnostic laboratories and controversial observations of thesetechniques have been reported (Azumi et al., 1990; Login et al., 1991;Azumi et al., 1991).

This invention relates to a method and apparatus for processing tissuesamples or other biological samples for a wide variety of purposes.Tissue samples are analyzed for many purposes using a variety ofdifferent assays. Pathologists often use histochemistry orimmunocytochemistry for analyzing tissue samples, molecular biologistsmay perform in situ hybridization or in situ polymerase chain reactionson tissue samples, etc. Often the sample to be analyzed will be frozenor embedded in paraffin and mounted on a microscope slide. A typicalimmunocytochemistry assay requires a series of many steps. Theseinclude: obtaining a tissue sample such as from a biopsy, fixing thetissue in formalin, processing the tissue overnight, embedding thetissue in paraffin, cutting serial sections and mounting on microscopeslides and drying. These steps are followed by steps to deparaffinize(treatments in xylene, ethanol and water), and finally the reaction canbe performed on the tissue which has been mounted on the slide.Typically a series of solutions including reagents such as antibodies,enzymes, stains, etc., is dropped onto the slide, incubated, and washedoff. Finally the sample may be viewed under the microscope. Clearlythere are many individual steps involved and each step takes time. Thecurrent invention shortens the time for each step to be completed, andtherefore shortens the time for the analysis of the tissue sample.

At present, two procedures are generally used in preparing specimens oftissue for microscopic examination. In one procedure a specimen isfrozen, cut and mounted on a slide in an elapsed time of about 15minutes. This so-called “frozen-section” procedure has the advantage ofenabling a rapid histological diagnosis to be made from the specimen,and it is frequently employed in situations where a diagnosis isnecessary while a patient is on an operating table. The procedurepossesses certain disadvantages in that the prepared slide does notpossess the uniformity of quality of morphology prepared by othermethods. Moreover, it is technically more difficult for serial sectionsof the same specimen to be examined by this procedure, and extremecaution must be exercised in cutting the specimen in order to ensure asufficiently thin section and to avoid the possibility of damagingdetails of the specimen. The most serious objection to using the frozensection procedure is the necessity of preparing all the slides requiredfor special stains and/or consultation and teaching purposes while thetissue is in the initial frozen state. If the tissue is thawed andrefrozen for sectioning, it is severely damaged. Thus, when thefrozen-section procedure is used in emergency situations, it iscustomary for another portion of the tissue specimen to be processed inthe manner described hereinafter in order to have tissue available foradditional sections if further examination becomes necessary.

In the other procedures, a slide of relatively high quality ofmorphology is produced when a section of the specimen is mounted in ablock of paraffin; however, the time required to process a specimen oftissue for mounting in paraffin is on the order of 24-48 hours ascompared with the minutes required to process a specimen by thefrozen-section procedure. In the preparation of paraffin slides, aspecimen of tissue is immersed initially in a fixing agent. The fixedspecimen is then immersed in a dehydrating agent, and afterward thespecimen is immersed in a clearing agent. Finally, the cleared specimenis immersed in a bath of paraffin which impregnates the specimen andpermits it to be sliced into thin sections for subsequent mounting ontoslides. Because of the length of time required to prepare specimens bythis process, it is customary for hospital laboratories to beginprocessing the specimens late in the afternoon after surgeons haveobtained specimens from their patients. The processing continues throughthe night, and slides of the specimens are available for microscopicexamination the next morning. Although the slides produced according tothis procedure are of higher quality of morphology than those producedby the frozen-section technique, the length of time required to processspecimens is too great to enable this procedure to be used in situationswhere time is of the essence.

In the conventional histopathology laboratory, specimens of tissuereceived from surgery or autopsy are trimmed and preserved in smallcontainers of formaldehyde. The specimens are processed to remove water,and they are mounted in blocks of paraffin which are cut into thinsections. The thin sections are floated on water to enable them to betransferred to slides, and the sections are securely mounted on theslides when they are heated. Thereafter, the paraffin around the mountedsections is removed, and the sections are stained to ready them formicroscopic examination.

The ability to obtain rapid results, for example, during an on-goingsurgery, permits a microscopic examination and diagnosis of a tissuesample and thus, due to this examination, enables suitable surgicalsteps to be taken during the initial surgery without requiring afollow-up later surgical procedure.

With the foregoing in mind, it is the primary object of the presentinvention to provide an improved method for preparing specimens oftissue for microscopic examination.

As a further object, the present invention provides an improved methodby which tissue specimens of relatively high-quality of morphology canbe processed for microscopic examination in a minimum amount of time.

The publications and other materials used herein to illuminate thebackground of the invention or provide additional details respecting thepractice, are incorporated by reference, and for convenience arerespectively grouped in the appended List of References.

U.S. Pat. No. 3,961,097 teaches a method of using low frequencyultrasound (50 KHz) to reduce the time to perform biological processessuch as fixing tissue and impregnating it with paraffin. This patentteaches placing the sample in a small beaker of reagent to react withthe sample and then placing the small beaker into a larger beaker ofwater which is then irradiated with ultrasound. This method helped tolimit damage to the sample from the ultrasound treatment. This variesfrom the instant invention which places the transducer which producesthe ultrasound radiation within one inch of the sample. The instantinvention uses ultrasound of a high frequency to minimize damage to thesample.

U.S. Pat. No. 5,089,288 also discusses the use of ultrasound treatmentin the processing of tissue samples to impregnate them with paraffin.This method utilized a frequency range of 35-50 KHz. This is a lowerfrequency than the range of 100 KHz to 50 MHz employed by the instantinvention. The higher frequencies of the instant invention result inless biological damage than do the lower frequencies of the prior art'288 patent.

Chen et al. (1984) studied the effect of ultrasound treatment on therate of an immunoassay performed on a test strip and saw that thereaction was greatly accelerated in the presence of ultrasound. Theultrasound treatment was performed with an ultrasonic cleaner which hada nominal acoustic power output of 50 W at 50 KHz. Tests at variouspower (watts) were performed by varying the voltage to the sonicator.

Nishimura et al. (1995) teach a method for combining ultrasoundtreatment with a postfixation step in staining for lipid with osmiumtetroxide. The exact specifications as to intensity and frequency of theultrasound treatment are not disclosed. The ultrasonic treatment wasperformed using an ultrasonic cleaner. In general, ultrasonic cleanersproduce a frequency of 20-50 KHz.

Yasuda et al. (1992) disclose a method for tissue fixation whichincludes a combination of microwave treatment as well as ultrasonictreatment. Overlapping pulses of a few seconds of microwave andultrasound were administered for several cycles. The ultrasoundgenerator was set at a dose of 20 W/cm2 and a frequency of 40 KHz andwas operated at 25 V. To decrease the occurrence of cavitation which iscommonly caused by ultrasound treatment, the experiments were performedat 25 V instead of 100 V, tap water cooled to 0° C. was placed betweenthe cup of the ultrasound generator and the plastic container thatcontained the tissue blocks and fixative, in order to make the fixativecool and to make three layers (water, plastic and fixative) so that theultrasound energy would be reduced, and saponin or NP-40 was added tothe fixatives to reduce the surface tension of the tissue blocks.

Podkletnova and Alho (1993) utilized ultrasound to increase the rate ofperforming immunohistochemistry. Samples were placed in plastic tubeswhich were placed in an ultrasonic bath of cold water (12° C.) andtreated with ultrasonic irradiation for 0, 5, 10, 15, 20, 30 or 40seconds. The sonicator was operated as 220V/50 Hz; 180 W input, 90 Woutput, and the transducer produced a 40 kHz frequency.

A publication by Polonyi et al. (1984) teaches the use of ultrasound toaccelerate glutaraldehyde-osmium fixation of animal tissues. The use ofmedium intensities with low frequency (20 KHz) gave good results withwet tissue but damage was caused with dehydrated tissue. Consequentlythe authors adopted a method of using ultrasound during the fixation,washing, postfixation and saturating steps while performing thedehydrating steps without ultrasound.

A publication by Sinisterra (1992) discusses applications of ultrasoundto biotechnology. It teaches that high intensity ultrasonic waves breakthe cells and denature enzymes. Low intensity ultrasonic waves canimprove the mass transfer of reagents and products through a boundarylayer.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a diagram showing frequency generated vs. power of ultrasoundproduced by a transducer. In this diagram the power is strongest at afrequency around 5 MHz. At higher and lower frequencies the powerdecreases. The exact values obtained depend upon the transducer which isused, the characteristics of tissue or sample being treated, and thesolution or solvent which is used.

FIG. 2 is a schematic diagram showing a tissue in a buffer being treatedwith ultrasound. An ultrasound generator controls a transducer whichproduces the ultrasound waves. Three sensors, A, B and C, are shown. Acentral processing unit (CPU) controls the ultrasound generator as wellas records data from the sensors.

FIG. 3A illustrates a monitor feedback system. The sensor measures theultrasound intensity which passes through the sample and feeds this to aCPU which feeds back to regulate the ultrasound generator.

FIG. 3B illustrates a monitor feedback system in which sensors monitorand/or measure parameters, e.g., size, type, density, etc., of a sampleand feed data to a central processing unit which feeds back to anultrasound generator controlling a transducer producing ultrasound waveswhich irradiate a sample.

FIG. 4 illustrates a second type of monitor feedback system. This systemincludes 3 sensors. Sensor B monitors the ultrasound emission, sensor Amonitors a property of the sample, and sensor C monitors a property ofthe sample. Information from the sensors is fed to a CPU which feedsback to control the ultrasound generator as well as a heating andcooling system.

FIG. 5A is a schematic of a setup for performing fixation and processingof a tissue sample using ultrasound as part of the process. Anultrasound generator (US) produces ultrasound which irradiates a sample.One sensor monitors the ultrasound, and other sensors monitor the sampleas well as the solution/solvent. This data is processed and used by theCPU to automate changes in sample and/or solution or solvent. Data fromthe temperature sensor can be used to regulate the ultrasound output sothat the temperature does not get too high.

FIG. 5B is a schematic of one possible setup for performing fixation andprocessing of a tissue sample while using ultrasound as part of theprocess. It is similar to the setup of FIG. 5A but includes moreaspects. The ultrasound generator puts out high frequency, highintensity waves which can be of a single frequency or a widebandfrequency and can be continuous or pulsed. The transducer or transducerscan have one head or multiple heads. The sample can be rotated or thetransducers can revolve around the sample to aid in producing an evenultrasound field. This can be performed in one, two or three dimensions(1D, 2D and 3D).

FIG. 6A is a schematic of one possible system for performing andmonitoring a biological reaction using ultrasound. It is similar to FIG.5A but only one sensor is shown which is used to monitor the ultrasoundintensity. Also, the intensity of the ultrasound being used is lower inthis system.

FIG. 6B is a schematic of one possible system for performing andmonitoring a biological reaction using ultrasound. It is similar to FIG.9 but only one sensor is shown which is used to monitor the ultrasoundintensity which is lower in this system.

FIG. 7 represents a system showing four solutions, each in a differentcontainer. The complete system of tissue, ultrasound generator,transducer, sensors and CPU can be moved from one container to the next.This is preferably controlled by a robotic system which is not shown.

FIG. 8 illustrates an automated setup for fixing a tissue sample usingultrasound. Reagents from container 1 are pumped to a reaction chamber 2containing sample 3. A pump 4 pumps solution from chamber 2 to a wastereceptacle 5. A distributor 6 driven by motor 7 selects betweendifferent reagent containers such that different reagents can be pumpedthrough reaction chamber 2. Tissue sample 3 is placed into the reactionchamber 2 with or without tissue cassette 8. A cover 9 encloses thechamber. A central processing unit (CPU) 10 controls motor 7 and pump 4.The CPU also controls the temperature of reaction chamber 2 byregulating a heating and cooling plate 11 in contact with the reactionchamber 2. The CPU also controls an ultrasound generator 12 andregulates the frequency and intensity of ultrasound being produced. Thetransducers 13 emit ultrasound radiation and the sensors 14 send thedigitized information to the central processing unit 10. The tissuesample can instead be a membrane or some other type of sample which isplaced into the reaction chamber 2.

FIG. 9 illustrates a setup for performing in situ PCR hybridization withthe ability to assay the reaction fluid for success of the PCR. Theelements are similar to those shown in FIG. 8. Here a tissue sample 20is mounted on a slide 21 with the tissue sample 20 facing into thereaction chamber 2 (as shown in FIG. 8). Pump 22 routes some of thesolution being withdrawn from the reaction chamber 2 to a gel 23 whichwill be used to check for the presence of PCR products. FIG. 9illustrates the optional use of more than one distributor.

FIGS. 10A-10B show pictures of tissue samples which have been H&Estained using either the routine fixation and processing method (noultrasound) (FIG. 10A) or the new technique (with ultrasound) (FIG.10B).

FIGS. 11A-B show pictures of tissue samples which have undergone CD5staining using the routine fixation and processing method (noultrasound) (FIG. 11A) or the new technique (with ultrasound) (FIG.11B).

FIGS. 12A-B show tissue sections which have undergone in situhybridization with poly-A mRNA using either the routine fixation andprocessing method (no ultrasound) (FIG. 12A) or the new technique (withultrasound) (FIG. 12B).

FIG. 13 shows the results of RT-PCR amplification of β-actin mRNA usingthe routine fixation and processing method (no ultrasound) or the newtechnique (with ultrasound).

SUMMARY OF THE INVENTION

The present invention is directed toward a method of decreasing the timefor conducting histology or pathology study on tissue samples, e.g.,biological reactions, fixation, processing, embedding, deparaffinizing,and dehydration by applying ultrasound to the tissue during theseprocesses. The present invention discloses improved methods over theprior art. The invention is directed toward the unexpected feature ofspecific high frequency and high power ratings of ultrasonic treatmentwhich result in superior results over the prior art. The high frequency,wide-band and high power ratings used are as follows and are showngraphically in FIG. 1:

1) 0.1 to 1 MHz at a low power setting;

2)1 to 10 MHZ with a high intensity setting;

3) 10 to 50 MHZ with a low intensity setting.

It is important to understand that in the practice of the currentinvention more power equates to a shorter time from beginning to end foreach reaction step and additionally less destruction of tissue matter.The present invention is distinguished over the prior art in that theultrasonic transducer is placed relatively close to the tissue samplebeing processed. The prior art uses a system in which the multipiecetissue sample is immersed in a liquid bath wherein the liquid bath issubjected to ultrasonic energy. The prior art method (low frequencyultrasound) results in much more tissue damage. The present inventioncan be used in a variety of histological, pathological, immunologicaland molecular techniques.

The present invention is directed to using high intensity, highfrequency, nondestructive, wide-band ultrasound for tissue fixation andprocessing. The tissue must receive at least 10 W/cm² and fairly evendistribution of the ultrasound. High intensities of the lower frequencyultrasound result in higher cavitation and are more harmful than higherfrequencies.

The invention is directed to using a single high frequency or to usingwide-band frequencies (0.1-50 MHz) high intensity ultrasound. The exactfrequency and power to use to yield the best results depends on avariety of physical characteristics of the types of tissue being used.

The invention is directed to using ultrasound in conjunction with wellknown techniques to decrease the time required to perform thetechniques, including immunological reactions, hybridizations, tissuefixation and processing, reactions on membranes as well as in tissues.The intensity of ultrasound used depends on the particular procedure andwill be in the range of about 0.001-20 W/cm².

Another aspect of the invention is that the complete process can becontrolled and monitored by a feedback sensor-central processor unit(CPU)-ultrasound generation system. All of the steps of fixation andprocessing can be controlled by the CPU-ultrasound generation system.Use of a feedback sensor allows for optimization of the processes.

One aspect of the invention is the placement of an ultrasound transducerwithin 2 inches, preferably within 1 inch of the object being subjectedto the radiation. Multiple transducers may be used to direct ultrasoundto a single object.

The ultrasound being utilized can be either a continual lower intensitytreatment or can be pulses of higher intensity of lower frequency.

DETAILED DESCRIPTION OF THE INVENTION

The rates of biological processes such as reaction rates of staining,hybridization, immunostaining, etc., as well as processes such as fixingand imbedding tissue samples in paraffin can be increased by exposingthe sample or tissue to ultrasound during the process. But as notedabove, this unfortunately very often could be harmful to the biologicalsample. It has unexpectedly and fortuitously been discovered that thefrequencies and power ranges disclosed herein exhibit superior resultsand substantially no or less tissue damage than the prior art methods.Low frequency ultrasound is harmful to tissues because it can causecavitation. High frequency ultrasound is much less harmful.Unfortunately high frequency ultrasound is more easily absorbed bytissue. Nonetheless this can be overcome by using higher intensities ofultrasound in conjunction with the higher frequencies. Combining theproper frequency and intensity of ultrasonic treatment results inenhanced rates of reaction with no or minimal damage to the biologicalsample. The specific parameters depend upon the biological sample andthe specific process being performed.

Over the past 50 years tissue fixation and processing has changed verylittle. Although MW irradiation has been applied in histology for tissuefixation to accelerate techniques such as histochemical staining, IHC,and antigen retrieval over the past 10-30 years, standardization of MWtechniques and accurate monitoring of the procedures are still lacking(Login, 1998). Current methods of fixation and tissue processing arestill like “batch or bulk cooking”. Even the newly developed ultra-rapidMW/variable pressure tissue processor (Visinoni et al., 1998) uses the“batch or bulk method”. Fixing and processing of needle and endoscopicbiopsies from liver and stomach (small pieces of tissue) as well aslarge pieces of tissue from uterus and lung have been performed in thesame bulk cooker. Large tissue is under fixed and small tissue is overfixed resulting in uneven fixation and processing. This type ofvariation makes it very difficult to perform IHC, ISH, PCR and otherassays. In our preliminary studies, the prototype ultrasound apparatuswas designed very roughly. The frequency and intensity chosen in thisstudy were also not perfectly optimized. Nonetheless, the studies havegenerated excellent morphology with good protein and RNA preservation.No doubt, standardization of ultrasound-mediated high-speed tissuefixation and processing techniques and accurate monitoring of theprocedures should further decrease the time and improve the quality ofresults. This can be accomplished by controlling all steps of fixing andprocessing by means of a sensor-central processing unit (CPU)-ultrasoundgenerator feedback system. Ultrasound sensors monitor and determine howmuch ultrasound is absorbed by solution and/or tissue. The size, type,density, water content, and other information of the tissue are measuredby ultrasound sensors before fixing and processing. The sensors send alldigitized data information to the CPU. The CPU analyses the digitizeddata and sends parameters to the ultrasound generator that in responseemits the proper frequency and intensity of ultrasound for a measuredtissue. During processing, the changes of tissue size, thickness,density, water content, solvent content, paraffin content and otherinformation are accurately monitored and detected by sensors whichcontinuously send changed digitized data information to the CPU andrejustify the ultrasound generation system. This is similar to anacoustic force microscope or ultrasound scanner that sees inside tissuewhere a digital signal is converted to an image showing differences indensity. During fixation the density of the sample changes. The CPU canbe programmed to change the solution when the sample reaches a specifieddensity. Under this kind of emission-monitoring system, 1) the fixed andprocessed tissue is always in perfect or optimal condition; 2) theprocessed tissue avoids damage caused by too aggressive ultrasoundemission and over-fixation caused by the tissue remaining too long infixative; 3) individual tissue processing can be standardized and tissuefixation optimized to avoid “bulk cooking”, and 4) different sizes ofDNA or RNA probes will react with DNA or RNA targets or differentaffinities of antibodies will react with antigens under optimal matchconditions.

The preferred range of ultrasound frequency is in the range of 100 KHzto 50 MHz. A frequency in the range of 0.1 to 1 MHz can be used but onlyat a low power range of 0.1-25 W/cm² or else cavitation will occur. Useof 1 MHz to 10 MHz is less destructive than the lower frequencies andallows the use of higher power (20-100 W/cm²) which helps chemicalspenetrate into the tissues. Higher frequencies such as 10-50 MHzincrease the rate of reaction even further but tend to generate heat andat these higher frequencies it again is necessary to lower the power toabout 5-50 W/cm², here for the purpose of limiting the heat produced.

The process of combining ultrasonic treatment with well known biologicalmethods is further enhanced by placing the ultrasound treatment undercomputer control in which part of the process or the complete process iscontrolled by inputting various parameters concerning the techniquebeing performed, data concerning the tissue composition (e.g., bone,fat, etc.), size, etc., and by use of a sensor to monitor theultrasound.

The mechanism of the ultrasound fixation and processing methods has notbeen established. However, the basic principles of ultrasound mayexplain the process. Ultrasound has long been employed in such diversemedical fields as physical therapy, kidney and gall stone ablation, andmedical diagnostics. Abundant information exists about both itsbiological effects and potential toxicity (Miller, 1991). Manyphenomena. including cavitation, thermal effects, generation ofconvective velocities, chemical effects, biological effects, andmechanical effects, have been considered to play an important role inthe ultrasound-mediated enhancement of fixation and processing (Milleret al., 1996). We have utilized the well-known fact that intenseultrasonic waves traveling through fixative, solvent and tissue generatecavitations that enlarge and implode creating tremendous power. Theseextreme conditions provide an unusual chemical reaction which may resultin acceleration of hydration of methylene glycol to formaldehyde,acceleration of its binding to various amino acids, and acceleration offixing, dehydrating, clearing and impregnating tissue in an extremelyshort time. Based on the same principles of ultrasound, we have appliedultrasound to immunology and molecular biology for acceleratingreactions such as immunologic reaction, hybridization, washing anddeparaffinizing.

One aspect of the invention is the use of transducers with multipleheads. It has been found that use of a broad band of frequenciessimultaneously during the processes can improve the process. A singlehead is less efficient at producing a wide range of desired frequencies.To overcome this shortcoming each transducer can be fitted with multipleheads with each head on a transducer covering a different range offrequencies. FIG. 1 illustrates broadband, high-frequency,high-intensity ultrasound generated by a single transducer. Power isgreatest in the mid-range of frequencies with low power at the low andhigh frequencies. Low frequency ultrasound is destructive to tissue, butthis destructiveness decreases as the frequency is increased.Conversely, low frequency ultrasound produces only a small amount ofheat, but heat generation increases as frequency increases. The use ofbroadband (or wideband) ultrasound is useful because tissueheterogeneity and different solvents result in different requirementsfor the specific tissue and process being used. Consequently a systemwith the ability to generate a broad band is preferred although notrequired. Multiple transducers can also be used to achieve broadbandultrasound emission and to generate a desired power level at a desiredfrequency.

Another aspect of the invention is irradiating the sample from multipledirections. This can be accomplished in a variety of ways. A singletransducer can be moved relative to the sample either by rotating thesample or by revolving the transducer around the sample. Alternatively,several transducers can be used simultaneously with the transducers setout in, e.g., a circular pattern around the sample. If desired, multipletransducers can be set out in a 3-dimensional pattern about the samplerather than the 2-dimensional pattern of a circle or the 1-dimensionalpattern of a single transducer.

It is preferable to use a system which results in an even distributionof ultrasound energy throughout the reaction chamber. Use of a single,small transducer can result in uneven distribution of ultrasound and thepositioning of the tissue within the reaction chamber could have a largeeffect upon the results, especially if using a system without any sortof feedback to control the ultrasound generator. Such a system makes iteasy to overfix or underfix a sample. An even distribution of ultrasoundenergy throughout the reaction chamber is preferred. This may beachieved by use of multiple transducers set out around the reactionchamber, by use of a single, well designed transducer which will put outan even signal across the reaction chamber, and/or by use of a welldesigned reaction chamber.

A further aspect of the invention is the use of sensors to determine theprogress of the treatment such that it can be determined when to stopthe treatment. Preferably this will be computer controlled by connectingthe sensors and the transducers to a central processing unit. As atissue sample is fixed, the intensity of ultrasound reflecting from orpassing through the tissue changes. This change is detected by thesensors and fed into the central processing unit. When the signalreaches a preset level the central processing unit will shut off thetransducer producing the ultrasound reflecting from or passing throughthe tissue to that sensor. In this manner there is assurance that thetissue is neither undertreated nor overtreated with ultrasound. Theexact settings will depend on factors such as the type and thickness ofsample being treated and the frequency and power of ultrasound beingused. Such settings can be determined by one of skill in the art withsome initial trial and error testings. Once determined for a type oftissue and thickness of that tissue the value can be used for all futuresamples of that type and thickness and the frequency and power settingsused with no need of testing further. This holds true not only fortissue fixation but also for any other procedure for which ultrasound isbeing used.

The idea of measuring the intensity of reflected ultrasound or ofultrasound passing through the sample is similar to that of measuringultrasound to assay for cracks in metals or ceramics. This is well knownin the prior art of those fields although those fields are quitedifferent from the fields of pathology and molecular biology.

One setup for treating tissue with ultrasound is shown in FIG. 2. Anultrasound generator causes a transducer to emit ultrasound waves of adesired intensity and frequency, with a range of intensities andfrequencies being used if desired. Tissue is placed in solution togetherwith the transducer and is exposed to the ultrasound waves produced bythe transducer. Although it is quite feasible to simply use a transducerand a piece of tissue and to expose the tissue to ultrasound at adesired frequency and intensity for a specific length of time, themethod can be improved by including one or more sensors to follow thereaction, e.g., fixation, impregnation with paraffin, etc. Sensor A isused to monitor the intensity of the ultrasound which passes through thetissue. The intensity will change over time as the reaction, e.g.,impregnation with paraffin, proceeds. The signal from sensor A can befed into a central processing unit (CPU) which in turn regulates theultrasound generator and can be programmed to adjust the intensityand/or frequency of the ultrasound being produced by the transducer. Itcan be desirable to change the intensity and/or frequency as the processproceeds, e.g., as the tissue takes up more paraffin or as it becomesmore dehydrated, depending on the process being performed. A secondsensor (sensor B) can also be used if desired. Sensor B measures theultrasound intensity in the solvent or solution in which the tissue isplaced. It measures this in a region such that it measures the intensityof ultrasound which has not passed through the tissue. This effectivelyserves as a baseline measurement which will depend not only on thesignal produced by the transducer but also depends upon the specificsolvent or solution. Consequently it can be used to account for the useof different solvents or solutions. As with sensor A, the signalreceived by sensor B can be fed into a CPU which controls the ultrasoundgenerator. The use of a third sensor can also be accommodated. Forexample, sensor C can be used to monitor a physical parameter of thetissue during the processing of the tissue, e.g., sensor C can be usedto measure the temperature of the tissue. Again, this information can befed back to the CPU and be used to regulate the ultrasound generatorthroughout the time course of the process. For example, if thetemperature of the tissue was getting too high a signal could be sent todecrease the intensity of the signal, to alter the pulsing of thesignal, or to turn off the signal for a time until the temperaturedecreased to a specified temperature. Further sensors can be added tothe system as desired. Furthermore, one can use any one sensor withoutthe others, e.g., sensor C as described above to measure a physicalparameter could be used alone in the absence of sensors A and B. Also,as noted above, it is unnecessary to use any sensors although it ispreferable to do so because the feedback system enabled by the sensorshelps prevent overprocessing or underprocessing of the tissue.

Some examples of the above processes are shown as flow diagrams. FIG. 3Ashows a flow diagram for a system utilizing a single sensor. Anultrasound generator controls an ultrasound transducer which emitsultrasound of a desired frequency and power. The ultrasound passesthrough a tissue and is detected by a sensor. The sensor sends a signalto a CPU which analyzes the signal and, if desired, digitizes thesignal, and in accord with a program controls the output of theultrasound generator. FIG. 3B is a flow diagram of a system similar tothat shown in FIG. 3A except that the sensor measures a physicalparameter of the tissue sample, e.g., size, type, density, temperature,etc.

FIG. 4 shows a flow diagram of a system using three sensors. In thissystem an ultrasound generator causes a transducer to produceultrasound, some of which passes through a tissue sample and some ofwhich passes only through solution, e.g., a fixative or a solvent.Sensor A measures the ultrasound signal which passes through the tissueand feeds this signal to a CPU for analysis. Sensor B measures theultrasound signal which passes through the solvent/solution only andfeeds this signal to a CPU. Sensor C does not measure any ultrasoundsignal, rather it is used to measure a physical parameter of the tissueitself. Sensor C also feeds a signal to a CPU for analysis. The CPU (orCPUs if more than one is used) analyze the data, digitize it and feedback to control the output of the ultrasound generator in accordancewith a program. For example, the system can be programmed to shut offonce the tissue reaches a specified density or a specified size or itmay be shut off when the signal measured by sensor B in this examplereaches a specified intensity. Alternatively, the system can beprogrammed to adjust the intensity and/or frequency of the sound wavesproduced by the transducer or to adjust whether to give off a continuoussignal or a pulsed signal as well as adjusting the rate of pulsing if apulsed signal is used. For example, it is useful to alter the frequencyduring fixation of tissue, with a high frequency being used early and alow frequency being used later as the tissue becomes fixed.

A more general ultrasound system setup is illustrated in FIG. 5A. Anultrasound generator produces ultrasound which irradiates a sample.Sensors are present to measure the incident ultrasound intensity and canalso measure ultrasound intensity which passes through the sample, sizeof the sample, temperature of the sample, etc. Data from the sensorsfeeds into a central processing unit which can control the ultrasoundgenerator as well as control movement of samples into and out of areaction chamber, change solution or solvent within a reaction chamber,etc. Preferably when this is used for fixation and processing of atissue sample a high frequency >0.1 MHz and high intensity >5 W/cm² isused. An even field of ultrasound radiation is also preferred. FIG. 5Brepresents further options including use of either a single frequency ora wideband of frequencies of ultrasound radiation. The radiation can becontinuous or given in pulses. Transducers can have one head or multipleheads. To produce a more even field of radiation on the tissue sample,the sample can be rotated and/or the ultrasound transducers can berevolved around the sample.

FIGS. 6A and 6B outline setups for performing biological reactions.These are similar to what is shown in FIGS. 5A and 5B for fixation andprocessing of tissue samples. For biological reactions the intensity ofthe ultrasound radiation will be much lower and there is less need tomonitor physical parameters of the sample, although such can be done ifdesired.

The experiments described herein use high frequency and high intensityultrasound for tissue fixation. The ultrasonic apparatus used in thepresent study consists of an ultrasonic generator and a 1.6-1.7 MHzceramic transducer with an adjustable output intensity range from 1 to22 W/cm². The safety range of this high frequency ultrasound irradiationhas been tested with a variety of different tissue sections in severaldifferent solutions including saline, 10% neutral buffered formalin(NBF), different concentrations of alcohol, xylene and 60° C. paraffinusing a variety of different ranges of ultrasound intensity. Thestructure of a 5 μ tissue section mounted on a microscope glass slidecontinuously irradiated with 1.6-1.7 MHz ultrasound at 20 W/cm²intensity (actual intensity received by the tissue) in a variety ofbuffers or solvents for 10-20 minutes did not show any difference ascompared to an untreated neighboring serial section when viewed by lightmicroscopy. However, neighboring serial sections were destroyed whenirradiated in a 40 kHz ultrasound cleaning water bath for 10 seconds ina variety of buffers or solvents (Yasuda et al., 1992). These resultsindicate that high frequency and high intensity ultrasound irradiationis very safe for thin tissue sections.

Fresh surgical specimens were cut into 3-4 mm slices. One slice oftissue sample was step-by-step placed in NBF, steps of alcohol, xylene,and 60° C. paraffin and immediately irradiated by 1.6˜1.7 MHz ultrasoundat 20±3 W/cm² intensity (actual intensity received by the tissue). Otherslices of tissue samples were subjected to a standard fixation(overnight) and processing (overnight) as controls. Consecutive 4-5 μsections were cut from an ultrasound treated block and a control block.The sections from both blocks allow parallel comparison of morphology(hematoxylin and eosin staining (H&E)), protein (immunohistochemistry(IHC)) and RNA (mRNA in situ hybridization, (mRNA ISH)) preservation.The size of mRNA templates preserved by the ultrasound and routinemethods was further observed with RT-PCR.

The sections from ultrasound treated tissue were excellent in histologicappearance as compared with their routine fixation and processingcounterparts. The color balance in the H&E ultrasound sectionconsistently demonstrated slightly more eosinophilia on the cytoplasmand more intense nucleus staining than the routine method (FIGS. 10A-B).All ultrasound irradiated tissue blocks sectioned as well as controltissue blocks and no difference was detected in the sectioning andstaining process. No evidence of cavitation tissue injury was noted inthe ultrasound treated specimens under the conditions employed in thisstudy. Ultrasound treated tissue sections following protease or MWantigen retrieval pretreatment showed no disintegration ordeterioration. This indicates that tissue is fixed by formalin ratherthan by alcohol (dehydration only 10-15 minutes) according to Azumi'sexplanation (Azumi et al., 1990).

The distribution of IHC for CD45, CD20, CD3, CD5, Bcl-2, cytokeratin,kappa and lambda from routine or ultrasound treated tissue sections issimilar in this study. Several of the factors involved in the process offixation were found to affect immunoreactivity of the antibodies used inthis study. These include the duration and the speed of fixation andprocessing, and the duration and the concentration of primary andsecondary (2°) antibody incubation. A short incubation with primary/2°antibodies/ABC (10 minutes/5 minutes/5 minutes) gave poor stainingresults compared to the overnight incubation. However, this method gavethe best measurement to evaluate the condition of antigen preservation.The tissue from ultrasound irradiated fixation and processingsignificantly improved the immunoreactivity of the majority antibodies(CD3) in this study, and also dramatically reduced the incubation time.The requirement of concentration of primary antibodies (cytokeratin) forultrasound treated tissue also was reduced more 20-fold compared to theroutine treated tissue. Ultrasound high-speed fixed and processedtissues demonstrated the optimal fixation condition that was stained byCD5 even without MW antigen retrieval pretreatment (FIGS. 11A-B).

ISH is an excellent method for visualization and accurate detection of aspecific gene (e.g., oncogene, tumor suppressor gene or viral gene) inindividual, morphologically defined normal and neoplastic cells in bothfresh and archival tumor specimens with light microscopy. mRNA ISH isone of the best methods to evaluate the condition of tissue mRNApreservation (Weiss and Chen, 1991; Harper et al., 1992). Since the polyd(T) probe presumably hybridizes to polyadenylated sequences of RNA, itwould be expected that this probe would hybridize to the majority ofmRNA species—only 10-30% of mRNA lack the polyadenylated tail.Ultrasound high-speed fixed and processed tissue dramatically improvedthe total polyadenylated mRNA preservation more than 20-fold in theperiphery and more than 100-fold in the center of tissue as comparedwith the routine method (FIGS. 12A-B). As a check on the validity of thepoly d(T) used to detect mRNA, we performed parallel studies to detect aspecific mRNA, using the probes recognizing kappa immunoglobulin lightchain mRNA. The even distribution of kappa mRNA protected was found inthe tissue treated by the ultrasound method. However, in the tissuetreated with the routine method, the periphery and center showed unevendistribution, i.e., there was good fixation of the tissue at theperiphery and good preservation of mRNA at the periphery due toinactivation of RNAse in that region therefore showing good results, butthe more interior regions of the tissue were not well fixed and mRNA wasnot well preserved and showed poor results.

The mRNA ISH is only to examine the quantity of mRNA preservation in thetissue section. For assessment of the quality of mRNA samples generatedfrom routine or ultrasound fixed and processed tissues, RT-PCR wasperformed with two different sets of primer pairs to amplify β-actinmRNA which was then detected with ethidium bromide (Fend et al., 1999a;Ben-ezra et al., 1991; Foss et al., 1994; Krafft et al., 1997). With thetwo sets of primer pairs used in this study, the expected sizes of thecDNA amplification products were 156 bp and 548 bp. The routine methodand the ultrasound treated FFPE tissue block (FFPE blocks were stored atroom temperature for 5 to 6 weeks until used) samples yieldedextractable RNA using the TRIzol LS technique (Krafft et al., 1997). RNAyields were similar. Fixation and processing by the routine methodrequired 32 hours and yielded 0.17 μg/μL of RNA with an A₂₆₀/A₂₈₀ of1.536. Using the ultrasound method, fixation and processing required onthe order of 1 hour and yielded 0.19 μg/μL of RNA with an A₂₆₀/A₂₈₀ of1.655.

RT-PCR of mRNA from both the routine method and the ultrasound treatedtissue blocks produced a PCR 156 bp product, with the samples treatedwith ultrasound producing a comparatively stronger band. Shorterproducts were amplified more efficiently than longer products, butP-actin mRNA fragments of 548 bp were successfully amplified from theultrasound treated tissue block (FIG. 13). No 548 bp signals wereobserved after amplification from routine FFPE tissue block and negativeRT controls (no addition of reverse transcriptase). It has been reportedthat it is difficult to amplify any mRNA larger than 200 bp in FFPEtissues (Fend et al., 1999a; Ben-ezra et al., 1991; Foss et al., 1994;Krafft et al., 1997).

The results of this study demonstrate that the ultrasound-fixing and-processing method provides excellent morphologic detail as well asexcellent preservation of a variety of protein antigen and mRNA withinone hour. It shows that ultrasound energy can have an important role inrapidly fixing and processing biologic samples for quick diagnosis,immunology and molecular biology study. The ultrasound method providescertain advantages over the routine method for performing IHC for someof the antigens evaluated in this study. For example, tissues fixed andprocessed by the ultrasound method 1) did not require MW antigenretrieval for the detection of CD5, 2) allowed the IHC to be completedwithin 20 minutes, and 3) allowed use of a very low concentration ofantibody (high affinity antigen-antibody reaction). Furthermore, RNA isvery unstable in tissue once removed from the body. High levels ofendogenous RNase must be inactivated before RNA and mRNA degradation.Demonstration of RNA preservation in this study shows that the RNaseenzymatic activity was sufficiently inactivated within 10 to 15 minutesby ultrasound fixation and processing (dehydration in ethanol, clearingin xylene and infiltration with paraffin for 15 minutes each) in 45minutes. The results show that the ultrasound method provides a highquantity of mRNA preservation in the periphery and in the center oftissue blocks used for ISH, and a high quality of mRNA preservation byRT-PCR. Several possibilities may be responsible for the differencesobserved in the quantitative mRNA ISH and qualitative yields of RT-PCRproducts between these two methods. First, routine fixation requires along time (12-18 hours), whereas ultrasound fixation occurs in less than10-15 minutes. The longer exposure of the fresh tissues to the fixativebefore embedding in paraffin may result in RNA degradation fromendogenous RNases, resulting in a smaller size of amplifiable RNA.Second, although inactivated RNases are reversible within 24 hours, thedegree of RNase inactivation, cross-linking reaction, and the rate offixative penetration into the tissue may be time dependent in theroutine method. Third, the presence of residual fixative in theextracted cells may affect the efficiency of the RT or PCR reaction,resulting in a reduced amount of amplicon produced with samples preparedby the routine method. All results suggest that the rapid ultrasoundmethod is a valuable technique to permit or archive optimal preservationof antigen proteins, as well as being useful for high quantity andquality RNA preservation.

The results of the instant study demonstrate that theultrasound-fixation method provides excellent morphologic detail as wellas excellent preservation of a variety of protein antigens and mRNA in amatter of minutes. It shows that ultrasound energy has an important rolein rapidly fixing biological samples for quick diagnosis and forimmunological and molecular biological studies. The optimal fixation isextremely important for preservation of natural structure of antigenproteins and inactivation of enzymatic activity. The protein enzymaticactivity must be inactivated very quickly and completely after biopsy.High levels of endogenous RNase must be inactivated before RNA and mRNAdegradation can occur.

Formaldehyde, when dissolved in water, quickly changes to methyleneglycol and its polymers with less than 0.1% of the formaldehyderemaining in solution although the methylene glycol penetrates tissuevery rapidly. Chemical cross-linking with tissue is a relatively slowprocessing “clock reaction” that depends on the release of freeformaldehyde by gradual hydration of methylene glycol to produceformaldehyde. The available free formaldehyde binds to various aminoacids (Fox et al., 1985). The reaction with tissue is largely reversibleover the first 24 hours, and the fixed surface tissue acts as a barrierto subsequent inward diffusion of fixatives. Ultrasound-energy overcomesthese disadvantages and achieves the optimal fixation. The range ofoptimal fixation (retaining the natural structure of antigen proteinswithout enzymatic activity) is relatively narrow, but is achieved withultrasound fixation.

Ultrasound fixation provided certain advantages over routine formalinfixation for the IHC of some of the antigens evaluated in this study.For example, tissues fixed by the ultrasound method did not require MWantigen retrieval for good detection of CD5 whereas the correspondingformalin-fixed specimens did require MW treatment. However, using pepsinor MW antigen retrieval pretreatment provided the best results for IHC(Tables 3-4). Furthermore, ultrasound fixed specimens were superior toroutine formalin fixed tissues for the IHC performed for short times(high affinity antigen-antibody reaction). These results indicate thatthe ultrasound fixation method of the instant disclosure is a valuabletechnique for achieving optimal preservation of antigen proteins whichare altered when the routine fixation method is used. The results alsoindicate that the natural structures of antigen epitopes fixed byultrasound retain their high affinity to react with antibody.

We have treated tissue samples with both IHC and mRNA ISH usingantibodies and probes (Harper et al., 1992) and compared the levels ofIHC and ISH signals obtained with two types of fixation procedures,these being routine fixation and fixation using ultrasound. Furthermore,three different times were used in the routine method. The results showthat ultrasound fixation provided even kappa immunoglobulin signallevels for both IHC and ISH which indicates that ultrasound can overcomefixed surface tissue which could otherwise act as a barrier tosubsequent inward diffusion of fixatives. Use of ultrasound results ineven fixation within the tissue blocks, and therefore results in evenpreservation of mRNAs. By contrast, as could be expected, 30 minutes or6 hours of routine fixation did not allow any ISH signal, and 22 hoursof routine fixation gave uneven results within the tissue blocks withbetter mRNA preservation in the periphery than in the center of thesections. However, the IHC results were not seriously affected by theroutine fixation. This further indicates that with the routine fixationmethod formaldehyde inhibition of RNase activity is more difficult thanpreservation of antigen epitopes. These phenomena have been observed byother researchers using perfusion and immersion fixation (Tournier etal., 1987).

The methods disclosed herein describe a novel technique for rapidlypreserving tissues for morphologic, biochemical and molecular studies.The ultrasound-mediated fixation and processing method allowpreservation of high quality morphology, proteins and mRNA from routineformalin fixation and processing. The technique is fast, simple, easy toperform, and versatile. The ultrasound fixed and processed tissue may beused for rapid IHC or ISH or for rapid clinical pathology diagnosis.High quality fixed tissue sections may be used for laser capturemicrodissection, mRNA extraction and PCR studies. High quality fixedtissue blocks may be used for high-throughput tissue microarray analysesof the DNA, RNA and protein targets for a large series of cancerresearch. The techniques described can be applied not only to tissuesections but also to assays being performed on a membrane (e.g.,Northerns, Southerns and Westerns), on DNA chips, or on any other typeof microarray.

Examples are set out below which detail specifics for a variety oftechniques including H & E staining, immunostaining, in situhybridization of nucleic acid, and reverse transcription polymerasechain reaction. These methods, apart from their being combined withultrasound treatment, are well known to those of skill in the art. Thepresent invention is further detailed in the following Examples, whichare offered by way of illustration and are not intended to limit theinvention in any manner. Standard techniques well known in the art orthe techniques specifically described below are utilized.

EXAMPLE 1 Tissue Fixation and Processing

A general procedure for preparing tissue fixed by NBF, processed andimbedded in paraffin using ultrasound as part of the process to decreasethe required time to prepare the sample for use follows. Specifics areset out in the Examples which follow the general procedure. Those ofskill in the art recognize that many variations can be made in thefollowing procedures and the values set forth in the followingprocedures and Examples are not meant to be limiting.

A) Fixation

Step 1: A fresh tissue sample is cut in a size range from 3 to 5 mmthick, preferably less than 5 mm thick.

Step 2: The tissue sample (a single piece) is immersed in a 10% formalinsolution or other acceptable fixative. Depending on the type of tissue,the sample is immersed from 5 to 30 minutes at 37° C., preferably for 15minutes. During the immersion the sample is subjected to ultrasonicenergy. When using a fixed power setting, the frequency of theultrasonic waves to be used depends upon the thickness and size of thetissue and is a frequency in the range of 0.1-50 MHz. Frequencies towardthe lower range are used for thick and large size tissue samples whereasthin and small sized samples use higher frequencies. When using a fixedfrequency the power rating is adjusted according to a size and thicknessof the tissue sample. Thick and large size tissues require use of higherpower and thin and small size tissues use lower power. The nature of thetissue is also important with tough tissue (e.g., uterine tissue orligament) requiring a lower frequency and higher intensity of ultrasoundwhereas soft tissue (e.g., fat, lung and liver) requires a higherfrequency and lower intensity of ultrasound.

B) Processing

Step 3: Dehydration: The tissue is first immersed in an 80% to 95%alcohol solution for 1 to 10 minutes at 37° C. The time is dependentupon the type and size of tissues and is preferably in the range of2.5-5 minutes at 37° C. During this step, the sample is subjected toultrasonic energy at the same or different frequency and power (e.g.,lower frequency and higher intensity) as used in Step 2. The sample isthen immersed in a 100% alcohol solution for 1 to 15 minutes at 37° C.,preferably 5-7 minutes at 37° C. During this step the sample issubjected to ultrasonic energy at the same or different frequency andpower (e.g., lower frequency and higher intensity) as in Step 2.

Step 4: Clearing: The tissue sample is immersed in a xylene solution for2 to 20 minutes at 37° C. The time is dependent upon the type and sizeof the tissue samples and is preferably 3 minutes at 37° C. During thisstep the sample is subjected to ultrasonic energy at the same ordifferent frequency and power (e.g., lower frequency and higherintensity) as used in Step 2.

Step 5: Infiltration: The tissue sample is immersed in a paraffinsolution for 2 to 20 minutes at 60° C. The time is dependent on the typeand size of the tissue samples, preferably 10-15 minutes at 60° C.During this step the sample is subjected to ultrasonic energy at thesame or different frequency and power (e.g., lower frequency and higherintensity) as used in Step 2.

C) Imbedding

Step 6: Imbedding: The tissue sample is imbedded in a paraffin blockwithout use of ultrasound and cooled to −10 to −20° C.

D) Deparaffinization and Hydration

During the following steps the tissue section sample is subjected toultrasonic energy for less than 2 minutes at a higher single frequencysetting and a lower power setting.

The tissue section sample is immersed in 4 changes of xylene solutionfor 10 seconds for each immersion. The tissue sample is immersed in twochanges of 100% alcohol for 10 seconds for each immersion. The tissuesample is immersed in two changes of a 95% alcohol solution for 5seconds for each immersion. The tissue sample is immersed in a 70%alcohol solution for 10 seconds. The tissue sample is washed withdistilled water or phosphate buffered saline (PBS) for 10 seconds.

E) Dehydration

During this procedure the tissue sample is subjected to ultrasonicenergy for a little longer than 1 minute.

The tissue sample is washed with distilled water for 10 seconds. Thetissue sample is immersed in a 70% alcohol solution for 10 seconds. Thetissue sample is immersed in two changes of a fresh 95% alcohol solutionfor 5 seconds for each immersion. The tissue sample is immersed in twochanges of a fresh 100% alcohol solution for 10 seconds for eachimmersion. The tissue sample is immersed in two changes of a freshxylene solution for 10 seconds for each immersion.

After imbedding a sample in paraffin and sectioning it, it can be usedfor a variety of techniques which are well known to those of skill inthe art. One common procedure is staining of the tissue section.Hematoxylin and eosin (H & E) is the most common staining procedure usedin pathology. Every case must have H & E staining for making apathologic diagnosis. Deparaffinized tissue section slides which are inslide holders are placed vertically into a staining dish with 500 mL ofhematoxylin solution for 10 seconds with ultrasound followed by washingwith running tap water in a staining dish for 5 seconds with ultrasound.The slides are placed in 95% ethyl alcohol for 5 seconds with ultrasoundand counterstained in eosin-phloxine solution for 10 seconds withultrasound. The samples are dehydrated and cleared using two changeseach of 95% ethyl alcohol, absolute ethyl alcohol, and xylene for lessthan one minute each in the presence of ultrasound. The ultrasound usedthroughout this procedure is in the range of 1-5 MHz and greater than 5W/cm².

The tissue which has been fixed and mounted can be used for any one ofseveral techniques such as staining, hybridization, etc. In performingthese techniques ultrasound treatment can be used in the steps used forthose treatments.

In performing ultrasound for postfixation treatments such as staining orhybridization, it is preferred that a specific frequency and power ofultrasound be utilized, although this will depend upon the exact tissue,size of the probe and treatment. It is preferred that each transducerhas a single head, each putting out a single frequency wave. Each headcan be a different frequency, preferably 1-5 MHz or 0.1-30 MHz. Usingthe prior art disclosure of 20 to 50 KHz results in undesirable effectsand causes cavitation and destruction in tissue samples. Each tissue(fat, bone, etc.) requires a different frequency. The present inventionallows the use of more power without destroying cells and thereforeequates to greater speed of reaction. The method of the presentinvention yields much larger RNA strands with the use of the specificultrasonic energy technique. Further, the present invention enables insitu hybridization and IHC results to be uniform throughout.

EXAMPLE 2 Ultrasonic Apparatus and Application

The ultrasonic apparatus used in the present study was specificallydesigned by the inventor and built by Bio-Quick, Silver Spring, Md. Itconsisted of an ultrasonic generator and a 1.6-1.7 MHz ceramictransducers (1.5 cm diameter) with adjustable output intensity range of1 to 22 W/cm². The tissue (only one sliced tissue at time), which wasirradiated by ultrasound in 200 mL of NBF, grades of alcohol, xylene and60° C. paraffin, was directly faced and aimed at the transducers. Thedistance between the transducers and irradiated tissue was within 3 cmto insure that the tissue received even and accurate ultrasound energy.The application of ultrasound was always continuous rather than pulsed.The tissue receiving ultrasound energy was monitored by means of a UW-3ultrasound wattmeter (Bio-Tek Instruments Inc., Winooski, Vt.). Thetemperature of the fixative inside the container was limited to 37° C.during the exposure to ultrasound.

EXAMPLE 3 Immunohistochemistry with Ultrasound

Tissue sections from each experiment were stained with a panel of sevenprimary antibodies (CD20, CD45, CD3, CD5, Bcl-2, kappa and lambda; Table1). The primary antibodies were demonstrated using the ABC method (Hsuet al., 1981; Chu et al., 1992) as follows: sections were deparaffinizedand taken through to 70% alcohol before being placed into water.Sections were then pretreated with/without antigen retrieval by MW (Shiet al., 1991) or pepsin (Chu et al., 1999) as required for each antibody(Table 1). Sections were rinsed in phosphate buffered saline (PBS) andincubated in primary antibody for 5-10 minutes at room temperature witha higher single frequency (1.6-1.7 MHz) setting and lower power (0.01-5W/cm²) setting of ultrasound. After three rinses in PBS for 5 secondswith ultrasound, sections were incubated in biotinylated second antibodyfor 2.5 or 5 minutes at room temperature with ultrasound. Sections wererinsed in PBS with ultrasound and treated with 0.3% hydrogen peroxidefor 1-2 minutes with ultrasound, followed by incubation in ABC complexfor 2 or 5 minutes with ultrasound. Antibody binding sites werelocalized using 3,3′-diaminobenzidine for 10 seconds with ultrasound andsections were lightly counter-stained with hematoxylin for 5 secondswith ultrasound. The ultrasound used throughout this Example was at1.6-1.7 MHz and 0.01-5 W/cm². The exact values depended upon thespecific antibody. A higher intensity was used for those that otherwisegave a higher background.

TABLE 1 CD Clone Dilution Pretreatment Supplier CD20/L26 Mono 1:400 NoneDako CD45/LCA Mono 1:250 None Dako CD3 Poly 1:500 Pepsin Dako CD5/4C7Mono 1:100 Microwave Novocastra Bcl-2 Mono 1:100 Microwave Dako KappaPoly 1:50,000 Pepsin Dako Lambda Poly 1:100,000 Pepsin Dako

EXAMPLE 4 In situ Hybridization with Ultrasound

Tissue sections were deparaffinized and prepared with enzymatic or MWantigen retrieval pretreatment before hybridization. A syntheticoligonucleotide probe directed against poly A⁺ mRNA and a probe for mRNAof kappa immunoglobulin were labeled with fluorescein isothiocyanate(FITC) (sequences provided by BioGenex, San Ramon, Calif.). FITC-labeledprobe was applied to the tissue sections which were then coverslippedand denatured at 100° C. for 5 minutes in a vegetable steamer. Theslides were cooled down and hybridized in the presence of ultrasound ata high single frequency with a low power setting at room temperature for10-60 minutes in the steamer. Sections were washed twice, 3 secondseach, in 2×SSC with ultrasound and then incubated for 10 minutes withmonoclonal mouse anti-FITC with ultrasound, followed by two washes inPBS with ultrasound, 3 seconds each. Biotinylated secondary antibody wasincubated with the tissue sections in the presence of ultrasound for 2-5minutes at room temperature, followed by two 3 second washes in PBS withultrasound. Streptavidin-biotinylated peroxidase and5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT)reagents were used in the presence of ultrasound. The ultrasound usedthroughout this Example was at 1.6-1.7 MHz and 0.01-5 W/cm². Theintensity used depended upon the probe length with a higher power beingused with longer probes.

EXAMPLE 5 Northern and Southern Hybridization with Ultrasound

Experiments were performed just as in Example 4 except that theexperiments were performed with the nucleic acid bound to a membranerather than being in situ.

EXAMPLE 6 Robotic System

In this system, illustrated in FIG. 7 the tissue sample as well as thetransducer and sensors are moved from one reaction chamber to the next.To fix a tissue sample, the tissue, transducer and sensors are allplaced into a first reaction chamber containing fixative. Aftertreatment with ultrasound in the fixative, a robotic system removes thetissue sample, transducer and sensors and moves them all to the nextreaction chamber containing ethanol. After treatment with ultrasound inthe ethanol is complete, the robotic system moves the tissue, transducerand sensors to a reaction chamber containing xylene. After treatment iscomplete in the xylene, the robotic system moves the tissue into areaction chamber containing paraffin at 60° C. The CPU is programmed tocontrol the ultrasound generator for each of these steps. Once thetissue is imbedded with paraffin, the fixed tissue is roboticallyremoved from the reaction chamber and surrounded with more paraffin tocreate a paraffin block.

EXAMPLE 7 Auto-Fixation and Processing Reactor

In Example 6, a robotic system was used to move the sample, transducerand sensors from one solution to the next throughout the fixationprocess. In this Example, illustrated in FIG. 8, the tissue, transducerand sensors remain fixed and the solutions are changed. The tissue isplaced in a reaction chamber through which fluids can be pumped. Meansfor heating and/or cooling the reaction chamber are also illustrated.Fluid or reagent is pumped into the reaction chamber and ultrasound isproduced by the transducer. The sensors monitor the reaction andfeedback through the CPU to control the ultrasound generator. Whenreaction with one fluid is complete, the fluid is pumped out as the nextfluid or reagent is pumped in. A distributor selects which fluid, or airor gas if desired, is pumped in. The used fluid is pumped out to a wastereceptacle or can be recycled if desired. Flow of fluids and reagentsthrough the reaction chamber can be continuous with a distributor,controlled by the CPU, changing which fluid/reagent enters the chamber.Alternatively, flow can be pulsed such that the chamber is filled, flowis stopped or circulated as the reaction occurs, and then flow beginsagain with the next reagent/fluid after the reaction has finished. Afterthe paraffin is imbedded, the chamber will cool to −10 to −20° C., aparaffin block is formed and is ready to be cut.

EXAMPLE 8 Auto Reaction with a Membrane

This system is nearly identical to that of Example 9 except that inplace of a tissue sample a membrane with bound sample (e.g., nucleicacid or protein) is placed into the reaction chamber. This can be usedfor Northern, Southern and Western blots, ELISA, etc. Furthermore, inplace of a membrane, a chip such as a DNA chip or an immuno chip can beused.

EXAMPLE 9 Automated Immunohistochemistry

This system is very similar to that of Example 7. Here a slide with atissue sample mounted on it is used in place of a tissue or a tissuesection in a reaction chamber. As illustrated (FIG. 9) the tissue isbelow the slide and above the transducer with a channel between theslide and the transducer. Heating and cooling elements can also beincluded. Solutions and reagents are passed through the channel suchthat they contact the tissue sample. The extent of reaction in thepresence of ultrasound treatment is measured by time or, if sensors arepresent, by feedback from the sensors. As each reaction is completed achange of solution/reagents occurs. Each fluid is pumped to a wastereceptacle or recycled.

EXAMPLE 10 Automated in situ Hybridization

This system is very similar to that of Example 9 except that in situhybridization is performed rather than immunohistochemistry.

EXAMPLE 11 In situ PCR Hybridization

This Example uses a setup very similar to that described in Example 9.The difference is an added pump which can be used to remove samples ofamplification fluid as the amplification is occurring. This is shown inFIG. 9. If desired, this added pump can deliver sample to a gel. Thiscan be performed at time points during the amplification if desired orsimply at the end of a specified time period. As the in situ PCR(polymerase chain reaction) is occurring, amplification occurs in thepresence of ultrasound and then hybridization occurs to nucleic acidwithin the cells of the tissue. Some of the amplified product stays inplace in the cells, but a large percentage of amplified product washesoff the tissue and into the solution. By sampling the solution it ispossible to determine whether the in situ PCR is working properly bymeasuring the size of the products being formed. If it does workproperly then the amplified nucleic acid product will appear in thesolution. Samples of the solution can be run on a gel and stained orautoradiographed to determine if a band of nucleic acid of the expectedsize has been produced. If the desired band is seen, the PCR has workedand it is worth continuing the workup of the in situ hybridization. Ifno band is seen on the gel then no amplification occurred and furtherworkup of the in situ hybridization will not be performed.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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What is claimed is:
 1. A method for fixing or processing a tissue samplecomprising exposing said tissue sample to ultrasound of a frequency ofat least 100 Khz wherein said ultrasound is produced by an ultrasoundtransducer.
 2. The method of claim 1 wherein only a single piece of saidtissue sample is placed into said transducer.
 3. The method of claim 1wherein said frequency is in the range of 100 KHz to 50 MHz and whereinsaid frequency is a single frequency or a wideband frequency.
 4. Themethod of claim 1 wherein two or more ultrasound transducers are used toproduce ultrasound.
 5. The method of claim 1 wherein one or moreultrasound transducers are used to produce an ultrasound field such thatat least a portion of said tissue sample receives ultrasound of auniform frequency and a uniform intensity.
 6. The method of claim 1wherein said transducer comprises only one head.
 7. The method of claim6 wherein said head is capable of emitting a wideband frequency.
 8. Themethod of claim 6 wherein said head is capable of emitting a singlefrequency or a wideband frequency.
 9. The method of claim 1 wherein saidtransducer comprises multiple heads.
 10. The method of claim 9 whereinone or more of said multiple heads are capable of emitting a widebandfrequency.
 11. The method of claim 9 wherein one head on a singletransducer produces a frequency different from a frequency produced by asecond head on said single transducer.
 12. The method of claim 9 whereinone head on a single transducer produces a range of frequencies and arange of intensities different from a range of frequencies and a rangeof intensities produced by a second head on said single transducer. 13.The method of claim 4 wherein each of said transducers produces anultrasound frequency different from an ultrasound frequency produced byat least one other transducer.
 14. The method of claim 4 wherein each ofsaid transducers produces a range of ultrasound frequencies and a rangeof ultrasound intensities different from a range of ultrasoundfrequencies and a range of ultrasound intensities produced by at leastone other transducer.
 15. The method of claim 13 wherein a range offrequencies is applied to said tissue sample.
 16. The method of claim 4wherein said transducers are arranged around said tissue sample in atwo-dimensional arrangement.
 17. The method of claim 4 wherein saidtransducers are arranged around said tissue sample in athree-dimensional arrangement.
 18. The method of claim 1 wherein saidtissue sample is rotated.
 19. The method of claim 1 wherein saidtransducer revolves around said tissue sample.
 20. The method of claim 1wherein said ultrasound is produced as a continuous signal.
 21. Themethod of claim 20 wherein said ultrasound is a single frequency in therange of 0.1-50 MHz.
 22. The method of claim 20 wherein said ultrasoundis a wideband frequency in the range of 0.1-50 MHz.
 23. The method ofclaim 1 wherein said ultrasound is produced in pulses.
 24. The method ofclaim 23 wherein said ultrasound is a single frequency in the range of0.1-50 MHz.
 25. The method of claim 23 wherein said ultrasound isproduced as a wideband frequency in the range of 0.1-50 MHz.
 26. Themethod of claim 23 wherein said pulses vary in frequency in the range of0.1-50 MHz.
 27. The method of claim 23 wherein said pulses vary inintensity.
 28. The method of claim 23 wherein said ultrasound isproduced as a continuous signal.
 29. The method of claim 28 wherein overtime said signal varies in frequency in the range of 0.1-50 MHz.
 30. Themethod of claim 28 wherein over time said signal varies in intensity.31. The method of claim 1 wherein said tissue sample receives ultrasoundof a power of at least 5 W/cm².
 32. The method of claim 1 wherein saidtissue sample receives ultrasound with a power in the range of 5-150W/cm².
 33. The method of claim 1 further comprising using one or moresensors to detect one or more parameters of reflected ultrasound whereinsaid parameters are selected from the group consisting of intensity andfrequency.
 34. The method of claim 33 wherein more than one type ofsensor is used.
 35. The method of claim 34 comprising an ultrasoundsensor and a sensor to measure temperature.
 36. The method of claim 33further comprising a central processing unit to monitor the sensorreadings.
 37. The method of claim 36 wherein said central processingunit controls said ultrasound generator.